Results Time-Course of Cell Growth and comet formation in DU 145 prostate cancer and TP 366 glioblastoma cells provoked by TMZ and TMZ-BioShuttle dilu-tion series Two different cell l
Trang 1International Journal of Medical Sciences
ISSN 1449-1907 www.medsci.org 2008 5(5):273-284
© Ivyspring International Publisher All rights reserved Research Paper
TMZ-BioShuttle – a reformulated Temozolomide
Waldemar Waldeck1, Manfred Wiessler2, Volker Ehemann3, Ruediger Pipkorn4, Herbert Spring5, Juergen Debus6, Bernd Didinger6, Gabriele Mueller1, Joerg Langowski1, Klaus Braun2
1 German Cancer Research Center, Division of Biophysics of Macromolecules, INF 580, D-69120 Heidelberg, Germany
2 German Cancer Research Center, Dept of Molecular Toxicology, INF 280, D-69120 Heidelberg, Germany
3 University of Heidelberg, Institute of Pathology, INF 220, D-69120 Heidelberg, Germany
4 German Cancer Research Center, Central Peptide Synthesis Unit, INF 580, D-69120 Heidelberg, Germany
5 German Cancer Research Center, Dept of Structural Analysis of Gene Structure and Function, INF 280, D-69120 Heidel-berg, Germany
6 University of Heidelberg, Dept of Radiation Oncology, INF 400, D-69120 Heidelberg, Germany
280, D-69120 Heidelberg, Germany Phone: +49 6221-42 2495; Fax: +49 6221-42 3375; e-mail: k.braun@dkfz.de
Received: 2008.08.18; Accepted: 2008.09.12; Published: 2008.09.15
There is a large number of effective cytotoxic drugs whose side effect profile, efficacy, and long-term use in man are well understood and documented over decades of use in clinical routine e.g in the treatment of recurrent glioblastoma multiforme (GBM) and the hormone-refractory prostate cancer (HRPC) Both cancers are insensitive against most chemotherapeutic interventions; they have low response rates and poor prognoses Some cytotoxic agents can be significantly improved by using modern technology of drug delivery or formulation We succeeded
to enhance the pharmacologic potency with simultaneous reduction of unwanted adverse reactions of the highly efficient chemotherapeutic temozolomide (TMZ) as an example The TMZ connection to transporter molecules (TMZ-BioShuttle) resulted in a much higher pharmacological effect in glioma cell lines while using reduced doses This permits the conclusion that a suitable chemistry could realize the ligation of pharmacologically active, but sensitive and highly unstable pharmaceutical ingredients without functional deprivation The re-formulation
of TMZ to TMZ-BioShuttle achieved a nearly 10-fold potential of the established pharmaceutic TMZ far beyond the treatment of brain tumors cells and results in an attractive reformulated drug with enhanced therapeutic in-dex
Key words: BioShuttle, Carrier Molecules; Drug Delivery; facilitated Transport; Glioblastoma multiforme (GBM); Reformula-tion, Temozolomide (TMZ)
Introduction
The medicinal treasures in the pharmacopoeia
worldwide, harboring multi-faced monographs,
whose pharmacologic potential albeit their therapeutic
limits are well known New formulations of
conven-tional cytotoxic drugs may open a door to a new
qual-ity of the pharmaceutical research This redesign of
“old fashioned” molecules to highly active
pharma-ceutical ingredients (API) could be a suitable practice
capable of improve the therapeutic index [1-4] In case
of malignant brain tumors especially in the
chemo-therapy of glioblastoma multiforme (GBM) the anti
cancer drug temozolomide (TMZ)
(8-carbamoyl-3-methylimidazo [5,1-d]-1,2,3,5-tetrazin-
4(3H)-one) has been well studied [5, 6] It has been
shown in recent phase III study, that a simultaneous
therapy with TMZ improves survival rates for patients
with GBM treated with radiotherapy [7] Encouraging data [8] give reason to expand the intervention with TMZ to difficult tumor types like prostate cancer Un-der clinical conditions TMZ was absorbed rapidly into the blood, and spontaneously decomposed at physio-logical pH to the cytotoxic methylating agent 5-(3-methyltriazeno)-imidazole-4-carboxamide
(MTIC) Its half-life and apparent oral systemic clear-ance values were 1.8 hours and 97 ml/minute/m2, however neutropenia and thrombocytopenia limited the tolerable application doses to 1000 mg/m2 The cytotoxicity of TMZ appears to be elicited through adduction of methyl groups to O6 positions of guanine (O6mG) in genomic DNA [9] followed by recognition
of this adduct by the mismatch repair system (MMR), which can mispair with thymine during the next cycle
of DNA replication [10, 11]
Trang 2The half-life of TMZ [12] in plasma and the
non-target-gene-specific alkylating mode of action can
lead to undesired adverse reactions, which could result
in discontinuation or interruption of therapy TMZ
therefore seems to be a good candidate for
reformula-tion, since our new TMZ derivatives could circumvent
these problems by retaining the high efficiency but not
the adverse effects of TMZ
The coupling of a peptide-based nuclear
localiza-tion sequence (NLS) leads to an active nuclear
target-ing minimiztarget-ing the above described handicaps (Drug
Design, Development and Therapy, in press) But due
to their higher molecular mass and their
phys-ico-chemical characteristics the transport of TMZ-NLS
peptide conjugates alone across the cellular membrane
is poor Therefore a transport molecule is needed so
that a sufficient concentration of pharmacologically
active molecules can reach their target side inside the
nucleus
Our efforts resulted in suitable ligation modes of
TMZ with a nuclear address peptide which in turn is
connected to carrier molecules For a better
under-standing a definition for “ligation” is given in
chemis-try the meaning of “ligation reaction” is the basis of the
Diels-Alder chemistry, which we focus on here
Such a ligation reaction should meet the
follow-ing criteria: (1) rapid course of the reaction, (2)
inde-pendent from solvent properties, (3) no side reaction
with other functional groups present in the molecules,
(4) without additional coupling-reagents, (5)
irreversi-ble chemical reaction characteristics, and (6) an
eco-nomical procedure
Our concept is based on the ‘Click Chemistry’ It’s
applications are increasingly found in all aspects of
drug discovery, ranging from clue finding through
combinatorial chemistry and target-templated in situ
chemistry, to proteomics and DNA research, using
Staudinger and Sharpless conjugation reactions
[13-16] In this regard the 1,3-dipolar cycloaddition
developed by Huisgen has to be considered as a 'cream
of the crop' [17]
Our ligation approach is based on cycloaddition
reactions via the pericyclic Diels Alder Reaction (DAR)
with ‘inverse-electron-demand’ (DARinv), which is a
modification of π-electron-deficient N-heteroaromatics
with electron-rich dienophils [18] The DARinv is, in
contrast to DAR, irreversible with the compounds we
used In this way the pharmaceutic TMZ was coupled
to the modularly structured carrier We called it
TMZ-BioShuttle During biological tests we reached a
dramatically increased efficiency in two different
tu-mor cell lines in the glioblastoma cell line TP 366 and
in the human prostate cancer cell line DU 145
The analyses of dilution series indicated for the application of the TMZ-BioShuttle that the spectra of the treatable tumor types could be extended
Materials and Methods
Synthesis of the TMZ-derivative
A 0.2 mol preparation with 42.7 mg 4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,
9-trien-9-carboxylic acid chloride and 67.4 mg
modi-fied tetrazine as well as 28 µl triethylamine were dis-solved in chloroform The reaction process runs un-disturbed and provides a defined product with the molecular weight (MW) m/e 513
Synthesis of the Boc-Lys(TCT)-OH
42 mg cyclooctotetraen and 44 mg maleic acid anhydride were resolved in chloroform and methanol
1 % The chemical reaction is described by Reppe [19]
Solid phase peptide synthesis of the BioShuttle transporter
For solid phase synthesis of the K(TCT)-NLS-S∩S-transmembrane transport peptide the Fmoc-strategy was employed in a fully automated multiple synthesizer (Syro II) [20] The synthesis was carried out on a 0.05mmol Fmoc-Lys(Boc)-polystyrene resin 1% crosslinked and on a 0.053 mmol Fmoc-Cys(Trt)-polystyrene resin (1% crosslinked) As coupling agent 2-(1H-Benzotriazole-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate (HBTU) was used The last amino acid of the NLS-peptide was incorporated as Boc-Lys(TCT)-OH Cleavage and de-protection of the peptide resin were affected by treat-ment with 90% trifluoroacetic acid, 5% ethanedithiol, 2.5% thioanisole, 2.5% phenol (v/v/v/v) for 2.5 h at room temperature The products were precipitated in ether The crude material was purified by preparative HPLC on an Kromasil 300-5C18 reverse phase column (20 × 150 mm) using an eluent of 0.1% trifluoroacetic acid in water (A) and 60% acetonitrile in water (B) The peptides were eluted with a successive linear gradient
of 25% B to 60% B in 40 min at a flow rate of 20 ml/min The fractions corresponding to the purified protein were lyophilized
Coupling of the transmembrane carrier - and the K(TCT)-NLS-Cys-module
The K(TCT)-NLS-C and the transport peptide were oxidized in an aqueous solution of 2mg/ml in 20% DMSO After five hours the reaction was com-plete The oxidation progress was monitored by ana-lytical C18 reversed-phase HPLC, and then the peptide was purified as described above The purified material
Trang 3was characterized with analytical HPLC and laser
de-sorption mass spectrometry in a Bruker Reflex II
Reagents and cell culture
Human glioblastoma (GBM) primary cells (TP
366) [21] and human prostate cancer cells (DU 145) [22]
were provided by the DKFZ division of Biophysics of
Macromolecules All cell lines were cultured in DMEM
(Gibco Cat No 12800) supplemented with 10% FCS
and maintained in culture at 37°C with 5% CO2
at-mosphere and 95% humidity
Chemotherapy treatment
Pure temozolomide (TMZ) was purchased from
Sigma-Aldrich, Germany (Cat No 76899) and the
material was subdivided into two parts for subsequent
processing One part was followed up and coupled to
the transporter molecules As a control, the second part
was dissolved in acetonitrile 10% (Sigma-Aldrich,
Germany) with a final concentration of 0.2%
acetoni-trile
DU 145 and TP 366 cells were seeded (1 × 105
cells/ml) in DMEM (control) and in DMEM containing
a dilution series of TMZ and of TMZ-BioShuttle from
50 to 6.25 µM respectively The behavior of the cells
was up to 6 days
Cell Cycle Analysis
The effects on the cell cycle distribution were
de-termined by DNA flow cytometry Flow cytometric
analyses were performed using a PAS II flow
cytome-ter (Partec, Muenscytome-ter, Germany) equipped with a
mercury vapor lamp (100 W) and a filter combination
for 2,4-diamidino-2-phenylindole (DAPI) stained
sin-gle cells From native probes the cells were isolated
with 2.1% citric acid/ 0.5% Tween 20 according to the
method for high resolution DNA and cell cycle
analy-ses [23] at room temperature Phosphate buffer (7.2g
Na2HPO4 × 2H2O in 100ml H2O dest.) pH 8.0
contain-ing DAPI for staincontain-ing the cell suspension was
per-formed Each histogram represents 30.000 cells for
measuring DNA-index and cell cycle For histogram
analysis, we used the Multicycle program (Phoenix
Flow Systems, San Diego, CA)
Cell viability
For detection of apoptotic cells and viability, a
FACS Calibur flow cytometer (Becton Dickinson
Cy-tometry Systems, San Jose, CA) was used with filter
combinations for propidium iodide For analyses and
calculations, the Cellquest program (Becton Dickinson
Cytometry Systems, San Jose, CA) was carried out
Each histogram and dot plot represents 10.000 cells
After preparation according to Nicoletti [24] with
modifications [25, 26], measurements were acquired in the logarithmic mode in Fl-3 and calculated by setting gates over the first three decades to detect apoptotic cells Dead cells are positive for propidium iodide and stained red, living cells remain unstained In the loga-rithmic histogram the positions of unstained living cells in a are in the first 2 decades the 3th decade con-tains cells with membrane damage, dead cells are placed in the 4th decade
Results
Time-Course of Cell Growth and comet formation
in DU 145 prostate cancer and TP 366 glioblastoma cells provoked by TMZ and TMZ-BioShuttle dilu-tion series
Two different cell lines originating from different tumor entities like the metastatic human prostate epi-thelium adenocarcinoma, herein referred as DU 145 (Figures 1-2) and TP 366 cells derived from human glioblastoma (Figures 3-4) were used to investigate the pharmacological effect of TMZ and TMZ-BioShuttle
To learn more about the DNA damage and cell death in the two different cell lines after treatment with TMZ as well as with TMZ-BioShuttle using the identical dilution series, we carried out comet assays in parallel probes (right columns of the figure 1 and 2) Basically the untreated cultures of DU 145 prostate cancer cells and TP 366 cells did not exhibit frag-mented DNA
DU 145 Prostate cancer cells
In order to determine the sensitivity of these cells
we used a dilution series in a range from 50 to 6.25 µM
of TMZ and TMZ-BioShuttle respectively
Two days after treatment with the TMZ final concentrations 6.25, 12.5 and 25 µM, the DU 145 cells seemed to be unfazed and no visual change in the phenotype could be observed under the light micro-scope Counting the corresponding cell numbers of-fered a hardly detectable decrease of the cell number (from 1.15 to 1.05 × 106 cells) if anything compared to the untreated control with 1.12 × 106 cells Only the probe treated with 25 µM TMZ revealed a decrease to 0.844 × 106 cells (Figure 1) The comet assay study in-dicated a higher sensitivity and exhibits fragmented DNA in the probes which were treated with 12.5, 25 and 50 µM TMZ The samples treated with 50 µM TMZ displayed a decrease of the cell number to 0.628 × 106 Additionally in the cell culture medium an increasing number of dead, clumped DU 145 cells could be ob-served
Trang 4Figure 1 Microscopical monitoring of the human prostate
cancer DU 145 cells 2 days after treatment with TMZ In the
comet column the scale bar represents 20 µm The microscopic
pictures were taken in phase-contrast, enlargement 200×
TP 366 glioblastoma cells
Light microscopical studies showed a reduced
cell population after treatment with 6.25 µM TMZ for 6
days which was increased further diminished under
two fold application doses (12.5; 25 µM) from 6.2 × 105
cells (untreated control) to 5.3; 4.8, and 2.1 × 105 cells In
the TP 366 cells the TMZ probe treated with 50 µM,
however, the cell population remained at the level of
the 25 µM treated cells (2.1 × 105 cells) and may reach a
saturation level
After 6 days treatment with 6.25 µM TMZ (figure
1 and 2) the TP 366 cells showed differently sized and
fragmented dead cells, sporadic comet structures and
shrunken nuclei In relation to the increased
applica-tion doses an increase of fracapplica-tionated DNA and shrunk
nuclei could be observed The TP 366 probe treated
with 50 µM TMZ exhibited sporadic comets and
al-most condensed nuclei The probes treated with
TMZ-BioShuttle displayed a deviant behavior The
6.25 µM TMZ-BioShuttle treated cells offer no DNA
fragmentation, but their nuclei seemed to be partly
swollen and diminished (figure 2) The latter fraction was increasing in accordance to the increased applica-tion doses In addiapplica-tion the total cell count of the TMZ treated samples was lower, compared to the cell number in the control and cells treated with TMZ-BioShuttle
The comet assay study detected no DNA frag-mentation in the untreated control TP 366 cells until
144 hours After application of TMZ-BioShuttle (6.25 µM) and more, much more DNA-fragments could be observed (figure 4) Indeed the use of higher applica-tion doses enhanced DNA fragmentaapplica-tions and the ra-tios of shrunk nuclei of TP 366 (figure 4 right column)
Figure 2 Microscopical monitoring of the human prostate cancer DU 145 cells 2 days after treatment with TMZ-BioShuttle In the comet column, the scale bars in the maps represent 20 µm The microscopic pictures were taken in phase-contrast, enlargement 200×
Trang 5Figure 3 Microscopic and comet assay studies of untreated
TP 366 cells and treated with various concentrations of TMZ;
the scale bar represents 20µm
Cell Cycle Studies
Here we present measurements of the cell
activi-ties like the cell cycle analysis using the flow
cytomet-ric method and a calculation of the percentage of cells
in different phases of the cell cycle These data were
obtained after treatment with TMZ and the
TMZ-BioShuttle in depency on the application dose
We determined the cell cycle behaviour of TP 366
glioma cells and DU 145 prostate cancer cells To
in-vestigate the influence of TMZ with and without
BioShuttle transporter we performed a dose-response
analysis of the cell cycle shown in Figures 5 and 6
TP 366 glioblastoma cells treated by TMZ and
TMZ-BioShuttle dilution series
Figure 5 exhibits the cell cycle distribution of TP
366 cells 6 days after treatment with increasing
con-centrations of TMZ and TMZ-BioShuttle is shown in
figure 5 The cell cycle distribution is signed as G1
phase, S phase and the G2/M phase These cells show
a diploid cycle (red coloured) The plot of the
un-treated control is demonstrated (figure 2, line 1) and
exhibits a G1 cell fraction of 86.4 % and a G2 cell
frac-tion 5.7 % Looking the TMZ treated cells the
contin-gent of cells in the S phase amounts to 7.8 % In com-parison to the S phase fraction of untreated control the percentage of S phase cells treated with 6.25 µM TMZ
is increased to 11.1 % The doubling of the TMZ dose to 12.5 µM results in a slightly decrease of the S phase fraction to 9.4 % The further increase of the TMZ concentration to 25 µM and 50 µM has barely influ-enced the amount of the cells in the S phase with 9.1 % and 9.8 % respectively Regarding the G2 phase we observed a dose dependent linear increase of the cell ratio A concentration of 6.25 µM depicted an increase
of cells from 5.7 % (control) to 10.2 % The TMZ dose of 12.5 µM results in a scarcely increase to 10.7 %, but the doubling of the TMZ concentration to 25 µM and fur-ther to 50 µM resulted in an intense increase of the cell fraction in the G2/M phase with 15.9 % and 21.3 % respectively The analysis of the dose dependent effect
on the G1 phase exhibited the following trend: the starting dose rate was 6.25 µM TMZ, doubling of the doses to 25 and to 50 µM showed a reciprocal propor-tionality to the percentage of G1 cells, 74.8% and 68.8% (figure 2, right column)
Figure 4 Microscopic and comet assay of untreated TP 366 glioblastoma cells, and 6 days after treatment with TMZ-BioShuttle; the scale bar represents 20µm Microscopic magnification is 200-fold in the phase contrast microscope
Trang 6The TMZ-BioShuttle treated TP 366 cells
pre-sented a cell cycle behaviour strongly differing from
the untreated control and from the TMZ treated cells
The probe treated with 6.25 µM evidenced a
re-duced cell fraction in G1 phase of 70.6% already The
TP366 cells treated with doses 12.5 and 25 µM indicate
G1 ratios of 57.9 and 37.6% respectively Especially the
data of the G2/M phase are demonstrative From 6.25
µM up to 25 µM we observed an increase of the ratio of
cells in G2/M from 19.1 %, via 28.6 % to 37.6 % In
comparison with the corresponding TMZ data, we
found a continuous G2/M enhancement with the
fac-tor 2 starting at 6.25 µM! to 25 µM and a facfac-tor 3 in the
probes treated with 12.5 µM
Due to the high amount of
dead cells the estimation of the
cell cycle distribution in TP 366 cells treated with 50
µM TMZ-BioShuttle was difficult to perform and to analyse It shows an increase of the cell fraction in the G1 phase to 68.8% which is identical to the corre-sponding probe treated with TMZ Consistently in-creased amounts of the S phase cells could be detected compared to both the S phase in the control and the corresponding probes treated with TMZ The trend of G2/M phase cells turned to the opposite direction In the S phase not definitive trend arose from the applied dose
Figure 5 The cell cycle distribution
in TP 366 glioblastoma cells dependent
on the applied concentration of TMZ
(right column) and TMZ-BioShuttle
(left column) 6 days after treatment
The axes of coordinates represent the
cell number; the abscissae represent the
corresponding DNA content The left
peak depicts the amount of cells in the
G1 phase; the area of the right peak
describes the ratio of cells in the G2/M
phase The area between both peaks
displays the amount of the cell fraction
residing in the S phase The insert
de-scribes the percentage of cells in the
phases of the cell cycle
Trang 7Figure 6 The cell cycle ratio of DU 145 prostate cancer cells dependent on the applied concentration of TMZ (right column) and TMZ-BioShuttle (left column) 2 days after treatment The axes of coordinates represent the cell number; the abscissae represent the corresponding DNA content The left peak depicts the amount of cells in the G1 phase; the area of the right peak describes the ratio
of cells in the G2/M phase The area between both peaks displays the amount of the cell fraction residing in the S phase
Trang 8Cell cycle behaviour of DU 145 cells after treatment
with TMZ and TMZ-BioShuttle dilution series
At a first glance the histograms reveal a lower
sensitivity of DU 145 prostate cells against TMZ
treatment compared to the results of TP 366 cells The
untreated DU 145 control exhibits a cell cycle
distribu-tion as follows: G1 – 58.5%; S phase – 29.8% and G2/M
phase 11.6% The ratio of TMZ treated cells in the
G2/M phase amounts to 12.8%; 15.4% and 23.5% and
is constantly increasing in correlation to the
applica-tion doses of 6.25; 12.5 and 25 µM
The percentage of the S phase cells after treatment
with the above described concentrations were almost
constant 29.5% and close to the control The cells in the
G1 fractions treated with increasing concentrations up
to 25 µM of TMZ presented a moderate but uniform,
continuous decrease from 58.5% (control), via 57.5%,
56.4%, to 48.6 % and then a strong decline to 29.8%
The DU 145 probe treated with 50 µM TMZ showed
22.3% G2/M phase cells and a dramatic increase of the
S phase cell fraction to 47.7%
The DU 145 cells treated with the TMZ-BioShuttle
under identical conditions offered a cell cycle
behav-iour which strongly differs from the TMZ probes The
percentage of cell fractions in G1 phase seemed to be
nearly constant 58.5% comparing the untreated control
versus the treated cells with concentrations 6.25µM
59.2 %and 12.5 µM 57.4% The concentrations of 25 and
50 µM gave rise to a marked decrease of the percentage
of cells from 45.5% to 20.7% in G1 which corresponds
to one-third of the related TMZ treated probe!
The relative amounts of S phase cells in the con-centration series with increasing TMZ-BioShuttle doses of 6.25, 12.5, 25, and 50 µM offered a reciprocal proportionality and showed a small but continuous decrease of cell number from 29.8% (control), via 28.8%, 28.1%, 27.1%, to 24.2% respectively It is im-portant to note that the latter result showed a strong reduction comparing 47.7% (TMZ) to 24.2% (TMZ-BioShuttle) which equates a degression of 50% The concentration series with increasing amounts of TMZ-BioShuttle indicated a direct concentration de-pendence and displayed a moderate increase of the cell number of DU 145 cells in the G2/M fraction from 11.6% (control) to 11.9% in the 6.25 µM probe via 12.5,
25 to 50 µM A strong rise of the S phase cells from 14.6% via 27.4% to 55.1% was shown Doubling the applied dose increased cell number in the G2/M phase mote than 100%! Additionally, the direct comparison
of the probe treated with 50 µM TMZ-BioShuttle with the corresponding TMZ probe of DU 145 cells showed
a dramatic increase in the percentage of cells in the G2/M phase (from 22.3% to 55.1%), revealing a G2/M block
Figure 7 The dose dependent effects of TMZ (C/D) as well as TMZ-BioShuttle (A/B) in the cell cycle behaviour of TP366 glioma (A/C) and DU 145 prostate cancer (B/D) cells ♦ G1phase; S phase; S G2/M phase
Trang 9Discussion
The number of old-fashioned cytotoxic drugs,
whose effectiveness is well understood, is multi-faced
However, if highly effective substances do not reach
their target site after application, what is their benefit?
Therefore questions about the bioavailability remain to
be answered, whereby the concentration in the
blood-stream is not meant here, but rather the concentration
directly at the site of pharmacological action, like the
genomic DNA in tumor cell nuclei
A substantial progress in the drug development
will be achieved by improvement of the delivery and
subcellular targeting of the drug as yet unappreciated
and meaningless
The BioShuttle delivery and targeting platform,
facilitating the transport of DNA derivatives into
liv-ing cells was described [27, 28] as well as transport of
diagnostics into the cytoplasm and nuclei of tumor
tissues [29, 30] There is no doubt that constructs like
the TMZ-BioShuttle, as an example, could play a
helpful role in the treatment of cancer The design of
such shuttles needs to incorporate features that reduce
undesired adverse reactions but maintains the efficacy
We selected the highly efficient chemotherapeutic
TMZ as a qualified candidate, since encouraging
re-sults in treatment of in brain tumors [31] remain
un-endorsed in the treatment of hormone-refractory
prostate cancer (HRPC) [32]
Parallel sets of experiments with TP 366 glioma
cells and DU 145 prostate cancer cells were carried out
and confirmed a lesser sensitivity of DU 145 prostate
cancer cells against TMZ treatment Whereas the TP
366 cells showed an increased DNA damage after TMZ
treatment up to a final concentration of 6.25 µM, the
DU 145 cells exhibited very few DNA damage
meas-ured by comet assay The increase of the application
dose however did not induce an increased number of
comets (figure 1) This phenomenon was already
documented in studies with ceramide-induced cell
death [33]
In DU 145 cells we compared the dose-dependent
application of TMZ and TMZ-BioShuttle of seeded
versus harvested cells after 48 hours In comparison to
the control (1.12) a linear inverse proportionality (1.15;
1.05; 0.844; and 0.628) to the dilution series of 6.25; 12.5;
25; and 50 µM TMZ was demonstrated
These data permit to assume a minimum of TMZ
application dose between 25 and 12.5 µM Dilution
series with 50 µM to 6.25 µM of TMZ-BioShuttle
showed an increase of shrunk nuclei (figures 2 and 4)
The TMZ-BioShuttle revealed the identical
subthresh-old in DU 145 cells but the quotient 0.414 at 50 µM suggests a higher pharmacological potential
The results achieved with both cell lines (TP 366 and DU 145) treated with the TMZ-BioShuttle differ in the expected augmentation of DNA comets: All cells were visibly swollen, but we could not detect an in-creased number of comets This suggests a deconden-sation of chromatin in the nuclei This happens when chromosomes are exposed to DNA replication inhibi-tion caused by failure to constitute compact chromatin areas during mitosis [34] It could be explained with the involvement of the transcription of genes express-ing histone regulatexpress-ing proteins responsible for forma-tion of the chromatin structure and for the compact package of DNA [35]
We investigated the cell cycle of TP366 after treatment of the glioblastoma cells with TMZ It inter-fered with the cell cycle and exhibited a decreased number of cells in the S phase fraction as shown 144 hours after TMZ-application when a contingent of the
S phase cells of 8% was detectable
The phenomenon of a reduced S phase (8% ver-sus control 11%) in the TMZ treated TP 366 cells is not contradictory to the common effects of alkylating agents which cause a retardation of the rate of cell di-vision [36] A possible explanation for the strongly decreased S phase cell number in the TMZ-BioShuttle treated probes would be the existence of a S phase cell cycle arrest as documented in a bimodal TMZ/ inter-feron-β (IFN-β) study [37] Our flow cytometry ex-periments also displayed a strong cell cycle arrest in the S phase [38] An interesting criterion for a pro-longed late S/G2 phase suggests the implication of the histone acetyltransferase 1 (HAT1) which participates
in recovering block-mediated DNA damages [39] The fact that the glioblastoma cells arrest in the G2/M phase after TMZ-treatment was documented by Hirose et al [40] and could be confirmed with the TP
366 cells Moreover the treatment with TMZ-BioShuttle resulted in a clearly increased G2/M phase of 72% It seems to be attributed to interactions of TMZ and TMZ-BioShuttle with the mitogen-activated protein (MAP) kinase p38α, which is activated by the mis-match repair system (MMR) and is responsible for the TMZ-induced G2/M block [40]
After cell exposure to the TMZ-BioShuttle for 144 hours, the observed strong increase of the G2 phase cells of TP 366 cells possibly originate from the already documented inhibition of the RNA and protein syn-theses, which are necessary for the successful comple-tion of G2 and the initiacomple-tion of mitosis These G2-arrested cells were found to be deficient in certain proteins that may be specific for the G2-mitotic
Trang 10transi-tion [41] The obstructransi-tion of the cell cycle process is
caused by the stop of this transition and results in the
decrease of the G1 phase cell fraction (26%) of the
TMZ-BioShuttle treated TP 366 cells which appear to
be consequence of their disability passing in the
mi-totic process
A sensitivity of DU 145 and TP 366 cells against
TMZ and a dramatically increased sensibility against
TMZ-BioShuttle is shown in figure 4 The present data
document a different cellular contumaciousness to
TMZ treatment A real effectiveness of the pure TMZ
in DU 145 cells could not be observed, whereas in
contrast clear cell killing effects, caused by
TMZ-BioShuttle, were detected
It is important to note that the TMZ-BioShuttle
treatment of DU 145 and the TP 366 cells redounds to
cell killing effects as shown in figure 4
As a general rule the different sensitivity of tumor
cells against chemotherapeutics is dependent on
mul-tiple mechanisms like mulmul-tiple drug resistance
sys-tems The relative low sensitivity of DU 145 prostate
cancer cells after treatment with TMZ is evident, and
the disappointing results of TMZ trials of prostate
cancer can be explained by the increased
O6-methylguanine-DNA methyltransferase (MGMT)
repair activity DU 145 cells show an increased MGMT
activity [42], associated with the decrease of the
pharmacologic effect after alkylating with TMZ alone
The increased MGMT expression and activity
corre-lates with the malignant phenotype [43] We regard the
potential utility of this epigenetic alteration as an
ap-propriate biomarker for prostate cancer [44] and an
important prognostic feature for the clinical outcome
in glioblastoma [45] The fact that die blood brain
bar-rier (BBB) no presents an hurdle for TMZ could explain
the higher pharmacological effects of TMZ in glioma
cells It is important in so far that the delivery and the
targeting of active substances play a decisive role and
must be further scrutinized The TMZ-BioShuttle could
be an appropriate candidate
The physico-chemical properties of TMZ which
have an impact on its bioavailability limit its
pharma-cological effectiveness Despite the benefit of the
con-comitant treatment with radiotherapy plus TMZ,
he-matologic toxic effects in patients treated with TMZ
are documented in multicenter studies by Stupp [46]
A TMZ-derivatization with targeting transporters
molecules and subcellular address components holds
tremendous potential to optimize treatment of
dis-eases Enhanced cellular delivery and active transport
of TMZ into the cell nuclei as site of pharmacological
action permit to expect lower application doses with
concomitantly decreased limiting side-effects
Our shuttle, designed for facilitating the rapid transport across cellular membranes, improved their own delivery and their own targeting under per-petuation of the pharmacological activity in cells and tissues while protecting cells in the bloodstream by omission of undesired side-effects like:
Strong suppression of the peripheral lympho-cytes result in discontinuation of therapy and prob-lems associated with systemic drug administrations, which are:
• even biodistribution of pharmaceuticals through-out the body;
• the lack of drug specific affinity toward a patho-logical site;
• the necessity of a large total dose of a drug to achieve high local concentration;
• non-specific toxicity and strong adverse side-effects due to high drug doses [47, 48]
The application of the TMZ-BioShuttle could minimize the handicap of TMZ on the therapy of pa-tients with brain tumors but the establishment of the TMZ-BioShuttle necessitates new ways for the synthe-sis Conditions which hampered the above postulated criteria like rapid and whole concurrent chemical re-actions in aqueous solution at room temperature for a proper chemical ligation of functional peptides could
be circumvented using the ‘inverse-electron-demand’
of the Diels Alder Reaction
Thus, TMZ-BioShuttle has been reformulated to decrease the toxic features in normal cells but retain the pharmacologic behavior in target cells; this was achieved as follows: coupling the amide group of the TMZ with a tetrazine which acts as dien-component and connects the NLS module with the tridecadien (TCT) component operating as dienophil
The presented publication shows that a proper chemistry contributes to the optimization of the pharmacological properties of the already efficient pharmaceutics like TMZ
The novel properties of the TMZ-BioShuttle could give reason to the extension to tumor types like pros-tate cancer, especially in hormone refractory situa-tions
Abbreviations
DAR: Diels-Alder-Reaction, GBM: Glioblas-toma Multiforme, HAT1: Histone Acetyltransferase 1, IFN-β: Interferon-β, MGMT: Methylgua-nine-DNA-Methyltransferase, NLS: Nuclear Localiza-tion Sequence, TCT: Tetracyclo-[5.4.21,7.O2,6.O8,11] 3,5-dioxo-4-aza-9,12-tridecadien, TMZ: Temozo-lomide